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Exome sequencing from a patient with neurological and developmental symptoms revealed two mutations in separate genes. One was a homozygous transition mutation that results in an in-frame, premature translational stop codon in the ZNF135gene predicted to encode a transcriptional repressor. Another mutation was heterozygous, a single nucleotide duplication in the KCNN2gene that encodes a Ca2+-activated K+channel, SK2, and leads to a translational frame shift and a premature stop codon. Heterologous expression studies, brain slice recordings, and coordination tests from a transgenic mouse line carrying the SK2 mutation suggest that it does not contribute to the patient’s symptoms. ZNF135is expressed in human brain and it is likely that the homozygous mutation underlies the human phenotype.

The plasticity of intrinsic excitability has been described in several types of neurons, but the significance of non-synaptic mechanisms in brain plasticity and learning remains elusive. Cerebellar Purkinje cells are inhibitory neurons that spontaneously fire action potentials at high frequencies and regulate activity in their target cells in the cerebellar nuclei by generating a characteristic spike burst-pause sequence upon synaptic activation. Using patch-clamp recordings from mouse Purkinje cells, we find that depolarization-triggered intrinsic plasticity enhances spike firing and shortens the duration of spike pauses. Pause plasticity is absent from mice lacking SK2-type potassium channels (SK2−/−mice) and in occlusion experiments using the SK channel blocker apamin, while apamin wash-in mimics pause reduction. Our findings demonstrate that spike pauses can be regulated through an activity-dependent, exclusively non-synaptic, SK2 channel-dependent mechanism and suggest that pause plasticity—by altering the Purkinje cell output—may be crucial to cerebellar information storage and learning.

Calcium ions are Nature's most widely used signaling mechanism, mediating communication between pathways at virtually every physiological level. Ion channels are no exception, as the activities of a wide range of ion channels are intricately shaped by fluctuations in intracellular Ca2+levels. Mirroring the importance and the breadth of Ca2+signaling, free Ca2+levels are tightly controlled, and a myriad of Ca2+binding proteins transduce Ca2+signals, each with its own nuance, comprising a constantly changing symphony of metabolic activity. The founding member of Ca2+binding proteins is calmodulin (CaM), a small, acidic, modular protein endowed with gymnastic-like flexibility and E-F hand motifs that chelate Ca2+ions. In this review, I will trace the history that led to the realization that CaM serves as the Ca2+-gating cue for SK channels, the experiments that revealed that CaM is an intrinsic subunit of SK channels, and itself a target of regulation.

Hippocampal-dependent synaptic plasticity and memory are modulated by apamin-sensitive small conductance Ca2+-activated K+ (SK) channels. Transgenic mice overexpressing SK2 channels (SK2+/T mice) exhibit marked deficits in hippocampal memory and synaptic plasticity, as previously reported. Here, we examined whether SK2 overexpression affects the encoding or retention of contextual memory. Compared with wild-type littermates, SK2+/T mice exhibited significantly less context-dependent freezing 10 min and 24 h after conditioning. Interestingly, this contextual memory impairment was eliminated if SK2+/T mice were permitted longer pre-exposure to the conditioning chamber. These data support converging evidence that SK2 channels restrict the encoding of hippocampal memory.

This summary article presents an overview of the molecular relationships among the voltage-gated potassium channels and a standard nomenclature for them, which is derived from the IUPHAR Compendium of Voltage-Gated Ion Channels.1 The complete Compendium, including data tables for each member of the potassium channel family can be found at http://www.iuphar-db.org/iuphar-ic/.

Small conductance, calcium‐activated potassium (SK) channels have an important role in determining the excitability and contractility of urinary bladder smooth muscle. Here, the role of the SK isoform SK3 was examined by altering expression levels of the SK3 gene using a mouse model that conditionally overexpresses SK3 channels (SK3T/T). Prominent SK3 immunostaining was found in both the smooth muscle (detrusor) and urothelium layers of the urinary bladder. SK currents were elevated 2.4‐fold in isolated myocytes from SK3T/Tmice. Selective suppression of SK3 expression by dietary doxycycline (DOX) decreased SK current density in isolated myocytes, increased phasic contractions of isolated urinary bladder smooth muscle strips and exposed high affinity effects of the blocker apamin of the SK isoforms (SK1‐3), suggesting an additional participation from SK2 channels. The role of SK3 channels in urinary bladder function was assessed using cystometry in conscious, freely moving mice. The urinary bladders of SK3T/Thad significantly greater bladder capacity, and urine output exceeded the infused saline volume. Suppression of SK3 channel expression did not alter filling pressure, threshold pressure or bladder capacity, but micturition pressure was elevated compared to control mice. However, SK3 suppression did eliminate excess urine production and caused a marked increase in non‐voiding contractions. The ability to examine bladder function in mice in which SK3 channel expression is selectively altered reveals that these channels have a significant role in the control of non‐voiding contractions in vivo. Activation of these channels may be a therapeutic approach for management of non‐voiding contractions, a condition which characterizes many types of urinary bladder dysfunctions including urinary incontinence.

Skeletal muscle hyperexcitability is characteristically associated with denervation. Expression of SK3, a small conductance Ca2+‐activated K+channel (SK channel) in skeletal muscle is induced by denervation, and direct application of apamin, a peptide blocker of SK channels, dramatically reduces hyperexcitability. To investigate the role of SK3 channels in denervation‐ induced hyperexcitability, SK3 expression was manipulated using a transgenic mouse that harbors a tetracycline‐regulated SK3gene. Electromyographic (EMG) recordings from anterior tibial (AT) muscle showed that denervated muscle from transgenic or wild‐type animals had equivalent hyperexcitability that was blocked by apamin. In contrast, denervated skeletal muscle from SK3tTA mice lacking SK3 channels showed little or no hyperexcitability, similar to results from wild‐type innervated skeletal muscle. However, innervated skeletal muscle from SK3tTA mice containing SK3 channels did not show hyperexcitability. The results demonstrate that SK3 channels are necessary but not sufficient for denervation‐induced skeletal muscle hyperexcitability. © 2002 Wiley Periodicals, Inc. Muscle Nerve 26: 817–822, 2002

Small conductance Ca2+-activated potassium (SK) channels underlie the afterhyperpolarization that follows the action potential in many types of central neurons. SK channels are voltage-independent and gated solely by intracellular Ca2+in the submicromolar range. This high affinity for Ca2+results from Ca2+-independent association of the SK α-subunit with calmodulin (CaM), a property unique among the large family of potassium channels. Here we report the solution structure of the calmodulin binding domain (CaMBD, residues 396–487 in rat SK2) of SK channels using NMR spectroscopy. The CaMBD exhibits a helical region between residues 423–437, whereas the rest of the molecule lacks stable overall folding. Disruption of the helical domain abolishes constitutive association of CaMBD with Ca2+-free CaM, and results in SK channels that are no longer gated by Ca2+. The results show that the Ca2+-independent CaM-CaMBD interaction, which is crucial for channel function, is at least in part determined by a region different in sequence and structure from other CaM-interacting proteins.

1Hyperexcitability in denervated skeletal muscle is associated with the expression of SK3, a small‐conductance Ca2+‐activated K+channel (SK channel). SK currents were examined in dissociated fibres from flexor digitorum brevis (FDB) muscle using the whole‐cell patch clamp configuration.2Depolarization activated a K+‐selective, apamin‐sensitive and iberiotoxin‐insensitive current, detected as a tail current upon repolarization, in fibres from denervated but not innervated muscle. Dialysis of the fibres with 20 mmEGTA in the patch pipette solution eliminated the tail current, consistent with this current reflecting Ca2+‐activated SK channels expressed only in denervated muscle.3Activation of SK tail currents depended on the duration of the depolarizing pulse, consistent with a rise in intracellular Ca2+due to release from the sarcoplasmic reticulum (SR) and influx through voltage‐gated Ca2+channels.4The envelope of SK tail currents was diminished by 10 μmryanodine for all pulse durations, whereas 2 mmcobalt reduced the SK tail current for pulses greater than 80 ms, demonstrating that Ca2+release from the SR during short pulses primarily activated SK channels.5In current clamp mode with the resting membrane potential set at −70 mV, denervation decreased the action potential threshold by ∼8 mV. Application of apamin increased the action potential threshold in denervated fibres to that measured in innervated fibres, suggesting that SK channel activity modulates the apparent action potential threshold.6These results are consistent with a model in which SK channel activity in the T‐tubules of denervated skeletal muscle causes a local increase in K+concentration that results in hyperexcitability.

We previously demonstrated that hIK1 is activated directly by ATP in excised, inside-out patches in a protein kinase A inhibitor 5–24 dependent manner, suggesting a role for phosphorylation in the regulation of this Ca2+-dependent channel. However, mutation of the single consensus cAMP-dependent protein kinase phosphorylation site (S334A) failed to modify the response of hIK1 to ATP (Gerlach, A. C., Gangopadhyay, N. N., and Devor, D. C. (2000) J. Biol. Chem.275, 585–598). Here we demonstrate that ATP does not similarly activate the highly homologous Ca2+-dependent K+channels, hSK1, rSK2, and rSK3. To define the region of hIK1 responsible for the ATP-dependent regulation, we generated a series of hIK1 truncations and hIK1/rSK2 chimeras. ATP did not activate a chimera containing the N terminus plus S1–S4 from hIK1. In contrast, ATP activated a chimera containing the hIK1 C-terminal amino acids His299–Lys427. Furthermore, truncation of hIK1 at Leu414resulted in an ATP-dependent channel, whereas larger truncations of hIK1 failed to express. Additional hIK1/rSK2 chimeras defined the minimal region of hIK1 required to confer complete ATP sensitivity as including amino acids Arg355–Ala413. An alanine scan of all non-conserved serines and threonines within this region failed to alter the response of hIK1 to ATP, suggesting that hIK1 itself is not directly phosphorylated. Additionally, substitution of amino acids Arg355–Met368of hIK1 into the corresponding region of rSK2 resulted in an ATP-dependent activation, which was ∼50‥ of that of hIK1. These results demonstrate that amino acids Arg355–Ala413within the C terminus of hIK1 confer sensitivity to ATP. Finally, we demonstrate that the ATP-dependent phosphorylation of hIK1 or an associated protein is independent of Ca2+.

In most central neurons, action potentials are followed by an afterhyperpolarization (AHP) that controls firing pattern and excitability. The medium and slow components of the AHP have been ascribed to the activation of small conductance Ca2+-activated potassium (SK) channels. Cloned SK channels are heteromeric complexes of SK α-subunits and calmodulin. The channels are activated by Ca2+binding to calmodulin that induces conformational changes resulting in channel opening, and channel deactivation is the reverse process brought about by dissociation of Ca2+from calmodulin. Here we show that SK channel gating is effectively modulated by 1-ethyl-2-benzimidazolinone (EBIO). Application of EBIO to cloned SK channels shifts the Ca2+concentration-response relation into the lower nanomolar range and slows channel deactivation by almost 10-fold. In hippocampal CA1 neurons, EBIO increased both the medium and slow AHP, strongly reducing electrical activity. Moreover, EBIO suppressed the hyperexcitability induced by low Mg2+in cultured cortical neurons. These results underscore the importance of SK channels for shaping the electrical response patterns of central neurons and suggest that modulating SK channel gating is a potent mechanism for controlling excitability in the central nervous system.

T cells express two different types of voltage‐independent Ca2+‐activated K+channels with small (SK) and intermediate (IK) conductance that serve important roles in the activation of T lymphocytes. In contrast to the IK channels from T lymphocytes which are upregulated upon mitogen stimulation, SK channels of Jurkat T cells, a human leukemic T cell line, are constitutively expressed even in the absence of mitogenic stimulation. We have used patch‐clamp recordings from transfected or injected mammalian cells to show that the cloned SK2 channel demonstrates the biophysical and pharmacological properties of the majority of K(Ca) channels in Jurkat T cells. The cloned and native channels are voltage‐independent, Ca2+‐activated, apamin‐sensitive, show an equivalent voltage‐dependent Ba2+block and possess a similar ion selectivity. In addition, we used the polymerase chain reaction to demonstrate the presence of SK2 mRNA in Jurkat T cells, whereas SK3 transcripts encoding the other cloned apamin‐sensitive SK channel were not detected. These data suggest that the voltage‐independent apamin‐sensitive K(Ca) channel in Jurkat T cells represents the recently cloned SK2 channel.

The activation of small-conductance calcium-activated potassium channels (SK) has a profound effect on membrane excitability. In hippocampal pyramidal neurons, SK channel activation by Ca2+ entry from a preceding burst of action potentials generates the slow afterhyperpolarization (AHP). Stimulation of a number of receptor types suppresses the slow AHP, inhibiting spike frequency adaptation and causing these neurons to fire tonically. Little is known of the gating properties of native SK channels in CNS neurons. By using excised inside-out patches, a small-amplitude channel has been resolved that was half-activated by ∼0.6μM Ca2+ in a voltage-independent manner. The channel possessed a slope conductance of 10 pS and exhibited nonstationary gating. These properties are in accord with those of cloned SK channels. The measured Ca2+ sensitivity of hippocampal SK channels suggests that the slow AHP is generated by activation of SK channels from a local rise of intracellular Ca2+.

HER-2/neuis a potent oncogene that predicts poor outcome when overexpressed in ovarian cancer. The SKOV-3 ovarian carcinoma cell line, one of the only models for HER2-driven ovarian cancer, expresses a major uncharacterized 8-kb alternative HER-2 transcript.

Skeletal muscle becomes hyperexcitable following denervation and when cultured in the absence of nerve cells. In these circumstances, the bee venom peptide toxin apamin, a blocker of small‐conductance calcium‐activated potassium (SK) channels, dramatically reduces the hyperexcitability. In this report, we show that SK3 channels are expressed in denervated skeletal muscle and in L6 cells. Action potentials evoked from normal innervated rat skeletal muscle did not exhibit an afterhyperpolarization, indicating a lack of SK channel activity; very low levels of apamin binding sites, SK3 protein, or SK3 mRNA were present. However, denervation resulted in apamin‐sensitive afterhyperpolarizations and increased apamin binding sites, SK3 protein, and SK3 mRNA. Cultured rat L6 myoblasts and differentiated L6 myotubes contained similar levels of SK3 mRNA, although apamin‐sensitive SK currents and apamin binding sites were detected only following myotube differentiation. Therefore, different molecular mechanisms govern SK3 expression levels in denervated muscle compared with muscle cells differentiated in culture. © 1999 John Wiley & Sons, Inc. Muscle Nerve 22: 742–750, 1999.

SK channels play a fundamental role in all excitable cells. SK channels are potassium selective and are activated by an increase in the level of intracellular calcium, such as occurs during an action potential. Their activation causes membrane hyperpolarization, which inhibits cell firing and limits the firing frequency of repetetive action potentials. The intracellular calcium increase evoked by action potential firing decays slowly, allowing SK channel activation to generate a long-lasting hyperpolarization termed the slow afterhyperpolarization(sAHP). This spike-frequency adaptation protects the cell from the deleterious effects of continuous tetanic activity and is essential for normal neurotransmission. Slow AHPs can be classified into two groups, based on sensitivity to the bee venom toxin apamin. In general, apamin-sensitive sAHPs activate rapidly following a single action potential and decay with a time constant of approximately 150 ms. In contrast, apamin-insensitive sAHPs rise slowly and decay with a time constant of approximately 1.5 s. The basis for this kinetic difference is not yet understood. Apamin-sensitive and apamin-insensitive SK channels have recently been cloned. This chapter will compare with different classes of sAHPs, discuss the cloned SK channels and how they are gated by calcium ions, describe the molecular basis for their different pharmacologies, and review the possible role of SK channels in several pathological conditions.

The nucleotide sequence of a complete cDNA copy of enkephalin precursor mRNA from human phaeochromocytoma is reported. The corresponding amino acid sequence shows that the precursor is 267 amino acids long and contains six interspersed Met-enkephalin sequences and one Leu-enkephalin sequence. Five of the seven enkephalins are flanked on both sides by pairs of basic amino acid residues. The precursor does not contain the sequences of the opioid peptides, dynorphin, α-neo-endorphin or β-endorphin.

Episodic ataxia (EA) is a rare inherited neurological disorder due to mutation in the voltage‐dependent Kv1.1 potassium channel. In nine unrelated families, a different missense point mutation at highly conserved positions has been reported. We have previously characterized six of the EA mutants. In this study, three recently identified mutations were introduced into the human Kv1.1 cDNA and expressed in Xenopusoocytes. Compared to wild type, T226A and T226M reduced the current amplitude by >95%, shifted the voltage dependence by 15 mV, and slowed activation and deactivation kinetics. Currents from G311S were ∼25% of wild type, less steeply voltage‐dependent and had more pronounced C‐type inactivation. These altered gating properties will reduce the delayed‐rectifier potassium current which may underlie the symptoms of EA.

weaver mice have a severe hypoplasia of the cerebellum with an almost complete loss of the midline granule cells. Recent genetic studies of weaver mice have identified a mutation resulting in an amino acid substitution (G156S) in the pore of the inwardly rectifying potassium channel subunit Kir 3.2. When expressed in Xenopus oocytes the weaver mutation alters channel selectivity from a potassium-selective to a nonspecific cation-selective pore. In this study we confirm by cell-attached patch-clamp recording that the mutation produces a non-selective cation channel. We also demonstrate that the cell death induced by weaver expression may be prevented by elimination of calcium from the extracellular solution as well as by coexpression with the wild-type Kir 3.2 allele, or other members of the Kir 3.0 subfamily. These results suggest that the weaver defect in Kir 3.2 may cause cerebellar cell death by cell swelling and calcium overload. Cells which express the weaver subunit, but which normally survive, may do so because of heteromeric subunit assembly with wild-type subunits of the Kir 3.0 subfamily.

The channel underlying the slow component of the voltage-dependent delayed outward rectifier K+current, IKs, in heart is composed of the minK and KvLQT1 proteins. Expression of the minK protein in Xenopusoocytes results in IKs-like currents, IsK, due to coassembly with the endogenous XKvLQT1. The kinetics and voltage-dependent characteristics of IsKsuggest a distinct mechanism for voltage-dependent gating. Currents recorded at 40mV from holding potentials between −60 and −120mV showed an unusual “cross-over,” with the currents obtained from more depolarized holding potentials activating more slowly and deviating from the Cole-Moore prediction. Analysis of the current traces revealed two components with fast and slow kinetics that were not affected by the holding potential. Rather, the relative contribution of the fast component decreased with depolarized holding potentials. Deactivation and reactivation, after a short period of repolarization (100ms), was markedly faster than the fast component of activation. These gating properties suggest a physiological mechanism by which cardiac IKsmay suppress premature action potentials.

Small conductance calcium-activated potassium channels show a distinct pharmacology. Some, but not all, are blocked by the peptide toxin apamin, and apamin-sensitive channels are also blocked by d-tubocurarine. Cloned SK channels (small conductance calcium-activated potassium channel) recapitulate these properties. We have investigated the structural basis for these differences and found that two amino acid residues on either side of the deep pore are the primary determinants of sensitivity to apamin and differential block by d-tubocurarine. Therefore, the pharmacology of SK channels compared with other potassium channels correlates with structural differences in the outer pore region. However, introduction of a tyrosine residue in the position analogous to that which determines sensitivity to external tetraethylammonium for voltage-gated potassium channels endows SK channels with an equivalent tetraethylammonium sensitivity, indicating that the outer vestibules of the pores are similar. The pharmacology of channels formed in oocytes coinjected with SK1 and SK2 mRNAs, or with SK1-SK2 dimer mRNA, show that SK subunits may form heteromeric channels.

Reproductive maturation and regulation is centrally orchestrated by gonadotropin-releasing hormone (GnRH). GnRH produced in the vertebrate hypothalamus acts on the pituitary to regulate gonadotropins. In nonplacental mammalian species, it has recently been shown that a second GnRH gene is expressed in mesencephalic cells. Here, we report the cDNA sequences and expression patterns for two distinct genes encoding the hypothalamic and mesencephalic GnRH forms in the brain of a placental mammal, the tree shrew (Tupaia glis belangeri). The novel mammalian GnRH form, designated here as [His5Trp7Tyr8]GnRH (often called chicken GnRH II), is expressed in neurons of the mesencephalon and is the first nonhypothalamic form to be isolated from a mammal. Its peptide sequence is identical to the form previously reported in fish, amphibians, reptiles, and birds, revealing that it has remained unchanged for 500 million years. In contrast, the sequences of the hypothalamic GnRH decapeptides vary by as much as 50% across vertebrate species. The remarkable sequence conservation of mesencephalic GnRH suggests that it has been highly constrained throughout evolution, perhaps indicating an important, conserved nongonadotropic role. The discovery and localization of two mRNAs encoding distinct GnRH forms in an advanced mammal suggest that other mammals, including primates, may also have a second GnRH gene with expression localized in the midbrain.

We have cloned from murine erythroleukemia (MEL) cells, thymus, and stomach the cDNA encoding the Ca2+-gated K+(KCa) channel, mIK1, the mouse homolog of hIK1 (Ishii, T. M., Silvia, C., Hirschberg, B., Bond, C. T., Adelman, J. P., and Maylie, J. (1997) Proc. Natl. Acad. Sci.(U. S. A.94, 11651–11656). mIK1 mRNA was detected at varied levels in many tissue types. mIK1 KCachannel activity expressed inXenopusoocytes closely resembled the Kcaof red cells (Gardos channel) and MEL cells in its single channel conductance, lack of voltage-sensitivity of activation, inward rectification, and Ca2+concentration dependence. mIK1 also resembled the erythroid channel in its pharmacological properties, mediating whole cell and unitary currents sensitive to low nmconcentrations of both clotrimazole (CLT) and its des-imidazolyl metabolite, 2-chlorophenyl-bisphenyl-methanol, and to low nmconcentrations of iodocharybdotoxin. Whereas control oocytes subjected to hypotonic swelling remained swollen, mIK1 expression conferred on oocytes a novel, Ca2+-dependent, CLT-sensitive regulatory volume decrease response. Hypotonic swelling of voltage-clamped mIK1-expressing oocytes increased outward currents that were Ca2+-dependent, CLT-sensitive, and reversed near the K+equilibrium potential. mIK1 mRNA levels in ES cells increased steadily during erythroid differentiation in culture, in contrast to other KCamRNAs examined. Low nanomolar concentrations of CLT inhibited proliferation and erythroid differentiation of peripheral blood stem cells in liquid culture.

The ATP‐sensitive K‐channel plays a central role in insulin release from pancreatic β‐cells. We report here the cloning of the gene (KCNJ6) encoding a putative subunit of a human ATP‐sensitive K‐channel expressed in brain and β‐cells, and characterisation of its exon‐intron structure. Screening of a somatic cell mapping panel and fluorescent in situ hybridization place the gene on chromosome 21 (21q22.1–22.2). Analysis of single‐stranded conformational polymorphisms revealed the presence of two silent polymorphisms (Pro‐149: CC‐CCand Asp328: GA‐GA) with similar frequencies in normal and non‐insulin‐dependent diabetic patients.

weavermice have a severe hypoplasia of the cerebellum with an almost complete loss of the midline granule cells. Recent genetic studies of weavermice have identified a mutation resulting in an amino acid substitution (G156S) in the pore of the inwardly rectifying potassium channel subunit Kir3.2. When expressed in Xenopusoocytes the weavermutation alters channel selectivity from a potassium‐selective to a nonspecific cation‐selective pore. In this study we confirm by cell‐attached patch‐clamp recording that the mutation produces a non‐selective cation channel. We also demonstrate that the cell death induced by weaverexpression may be prevented by elimination of calcium from the extracellular solution as well as by coexpression with the wild‐type Kir3.2 allele, or other members of the Kir3.0 subfamily. These results suggest that the weaverdefect in Kir3.2 may cause cerebellar cell death by cell swelling and calcium overload. Cells which express the weaversubunit, but which normally survive, may do so because of heteromeric subunit assembly with wild‐type subunits of the Kir3.0 subfamily.

Calcium-activated potassium channels are fundamental regulators of neuronal excitability, participating in interspike interval and spike-frequency adaptation. For large-conductance calcium-activated potassium (BK) channels, recent experiments have illuminated the fundamental biophysical mechanisms of gating, demonstrating that BK channels are voltage gated and calcium modulated. Structurally, BK channels have been shown to possess an extracellular amino-terminal domain, different from other potassium channels. Domains and residues involved in calcium-gating, and perhaps calcium binding itself, have been identified. For small- and intermediate-conductance calcium-activated potassium channels, SK and IK channels, clones have only recently become available, and they show that SK channels are a distinct subfamily of potassium channels. The biophysical properties of SK channels demonstrate that kinetic differences between apamin-sensitive and apamin-insensitive slow afterhyperpolarizations are not attributable to intrinsic gating differences between the two subtypes. Interestingly, SK and IK channels may prove effective drug targets for diseases such as myotonic muscular dystrophy and sickle cell anemia.

Inwardly rectifying K⁺ channel subunits may form homomeric or heteromeric channels with distinct functional properties. Hyperpolarizing commands delivered to Xenopusoocytes expressing homomeric Kir4.1 channels evoke inwardly rectifying K⁺ currents which activate rapidly and undergo a pronounced decay at more hyperpolarized potentials. In addition, Kir4.1 subunits form heteromeric channels when coexpressed with several other inward rectifier subunits. However, coexpression of Kir4.1 with Kir3.4 causes an inhibition of the Kir4.1 current. We have investigated this inhibitory effect and show that it is mediated by interactions between the predicted transmembrane domains of the two subunit classes. Other subunits within the Kir3.0 family also exhibit this inhibitory effect which can be used to define subgroups of the inward rectifier family. Further, the mechanism of inhibition is likely due to the formation of an “inviable complex” which becomes degraded, rather than by formation of stable nonconductive heteromeric channels. These results provide insight into the assembly and regulation of inwardly rectifying K⁺ channels and the domains which define their interactions.

Human reproduction is controlled by the hypothalamic–pituitary–gonadal axis laid down early in fetal development. Luteinizing hormone releasing hormone (LHRH), also termed gonadotropin releasing hormone (GnRH), is a decapeptide1,2and is a key molecule in this control circuit. It is produced by hypothalamic neurones, secreted in a pulsatile manner into the capillary plexus of the median eminence and effects the release of luteinizing hormone and follicle stimulating hormone from gonadotropic cells in the anterior pituitary3. The peptide may have further functions, including behavioural ones4, as LHRH or LHRH-like immunoreactivity has been found in gonadal tissue5,6, placenta7,8and the central nervous system9,10, and exogenously administered LHRH is shown to affect behaviour11,12. To investigate the biosynthesis of LHRH, we have now isolated cloned genomic and cDNA sequences encoding the precursor form of LHRH, the existence of which had been suggested from Chromatographic studies of hypothalamic8,13and placental8extracts. These DNA sequences code for a protein of 92 amino acids in which the LHRH decapeptide is preceded by a signal peptide of 23 amino acids and followed by a Gly-Lys-Arg sequence, as expected for enzymatic cleavage of the decapeptide from its precursor14–16and amidation17of the carboxy-terminal of LHRH.

A recombinant plasmid has been constructed which contains the mature protein coding region of the human serum albumin (HSA) gene. Bacteria containing this plasmid synthesize HSA protein under control of the E. coli trp promoter-operator. The DNA sequence and predicted protein sequence of HSA were determined from the cDNA plasmid and are compared to existing data obtained from direct protein sequencing. The DNA sequence predicts a mature protein of 585 amino acids preceded by a 24 amino acid “prepro’ peptide.